Turn to the PAG-E-SDS now!!!

[2810712]

SDS adds to mass as well as charge of the protein , to have the

separation only on the basis of molecular wt. after adding SDS the q/m

of all prots should be equal and as SDS also adds to molec. wt. of prot also, we should be able to get the original molecular wt. by comparing its mobility with that of a known sample molecular wt.

 

In SDS PAGE we use sodium dodecyl sufate to make the ratios of q/m s of two diff peptides nearly equal still keeping the difference between them the same. This tells that this ration is only important here... why???

 

 

I think the difference in the displacements of the two peps in the gel depends only of the ration of their q/ms. What is its expression???

 

DON'T mind Yggdrasil, if u r here...

 

Shrei

[Nashyboyo]

The mass/charge ratio is what separates proteins of different sizes. I can't think of the formula. The bigger the protein the more SDS associates with it thus more charge. More charge gives more mobility towards the anode and away from the cathode.

[2810712]

thnx for reply

 

we can't make definite statements about mol.wt.s of proteins without a formula...

 

 

 

 

hrushikesh

[Bluenoise]

we can't make definite statements about mol.wt.s of proteins without a formula...

hrushikesh

 

You also can't make one with a formula. Electrophoresis is a very variable form of measurement. Each run will go sligtly different due to the many factors involved that you really can't account for.

 

I'll give you the formula, but I don't really care to explain it, since it's fairly useless anyways.

 

ZeE = fu

 

The only way to make a real comparison is to run a set of proteins of standard weight along side your samples. Then estimate.

 

Alternatively you could plot the distance vs time fit it to this formula. However it is ONLY good for samples run on the same gel at the same time.

 

logM = a - bx

 

M= mass of protein.

x= distance travelled

a,b are constants individual to every gel.

 

enjoy.

[Nashyboyo]

Yeah that's right. You have to construct a calibration curve using the ladder to estimate the Mr of the protein(s).

[2810712]

Thanks for those formulae...today its fairly hard to understand and estimate

the errors so as to get correct forumula... and as the errors are variable with gels it seems fairly useless.

 

 

hrushikesh

[Yggdrasil]

In order to get an accurate molecular weight determination of a protein, you generally will not rely on only one technique. Proteins will often run at a different location on the gel than predicted for various reasons, so molecular weight calculation from SDS-PAGE can often be misleading. However, in addition to SDS-PAGE which measures the molecular weight of the denatured protein, techniques such as analytical gel filtration or analytical ultracentrifugation are used to measure the molecular mass of your protein in its native state, and can provide independent confirmation of the molecular mass determined using SDS-PAGE. However, if you just need a quick confirmation that your protein is of the correct size, SDS-PAGE provides a reasonable estimate of molecular weight.

[Bluenoise]

Or as always when in doubt just do mass spec. Though you should have a very pure sample

[2810712]

that helped frndz, informative... thanks alot.

 

 

 

hrushikesh

[Run]

The separating difference by SDS-PAGE is because of friction effects.

the proteins are separated in a mass dependent manner. If a constant force (F) would be applied on the proteins the low molecular mass (m) proteins would have a higher acceleration (a = F/m) compared to the high molecular mass proteins. However, not only the acceleration but also the force applied on the SDS coated proteins is mass dependent, since high molecular mass proteins carry more negative charged SDS (sodium dodecyl sulfate) (ratio 1.4:1). This results in a same velocity for all the different proteins. However, counting for the friction effects contributed by the gel, the heavy proteins are delayed more compared to the small proteins resulting in a mass dependent separation.